Empagliflozin reverses obesity and insulin resistance through fat browning and alternative macrophage activation in mice fed a high-fat diet

Figures

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  Figure 1. Empagliflozin (Empa) reduced weight gain and increased energy expenditure in mice fed a high-fat diet (HFD). (A) Design of study examining the therapeutic effects of Empa. (B) Body weights, (C) tissue weights, (D) oxygen consumption (VO2), (E) carbon dioxide production (VCO2), (F) respiratory exchange ratios (RERs), (G) energy expenditure and (H) rectal temperature of mice fed NC, an HFD and an HFD with Empa (HFD+Empa). Rectal temperatures were measured on three consecutive days between 09:00 and 10:00 hours using a microprobe thermometer. Data are presented as means±SEM, n=7–8. *p<0.05 and **p<0.01, respectively, for comparisons with NC-fed mice; #p<0.05 and ##p<0.01, respectively, for comparisons with HFD-fed mice. BAT, brown adipose tissue; Empa, empagliflozin; HFD, high-fat diet; NC, normal chow.

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  Figure 2. Empagliflozin (Empa) increased uncoupling protein 1 (Ucp1) expression in both brown (BAT) and white (WAT) adipose tissues. (A) BAT sections stained using H&E. Scale bars=100 µm. (B) Immunoblots of Ucp1 in BAT. (C) Levels of thermogenesis-related gene mRNAs in the BAT. (D) Immunoblots of Ucp1 in the inguinal (iWAT) and epididymal (eWAT) WAT. (E) Levels of browning-related gene mRNAs in the iWAT. Data are presented as means±SEM, n=7–8. *P<0.05 and **p<0.01, respectively, for comparisons with normal chow (NC)-fed mice; #p<0.05 and ##p<0.01, respectively, for comparisons with high-fat diet (HFD)-fed mice.

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  Figure 3. Empagliflozin (Empa) ameliorated high-fat diet (HFD)-induced hepatic steatosis and insulin resistance. Results of (A) glucose tolerance and (B) insulin tolerance tests. (C) Fed and fasting plasma insulin levels in mice fed normal chow (NC), an HFD and an HFD+Empa. (D) Immunoblots of phosphorylated Tyr1146 insulin receptor β subunit (p-IRβ), IRβ, phosphorylated Ser473 Akt (p-Akt) and Akt in the epididymal white adipose tissue (eWAT), liver and muscle of mice. (E) H&E-stained liver sections. Scale bars=100 µm. (F) Liver triglyceride levels, (G) mRNA levels of lipogenesis-regulated and fatty acid oxidation-regulated genes in the liver, and (H) liver TBARS contents in mice fed NC, an HFD and an HFD+Empa. Data are presented as means±SEM, n=7–8. *p<0.05 and **p<0.01, respectively, for comparisons with NC-fed mice; #p<0.05 and ##p<0.01, respectively, for comparisons with HFD-fed mice.

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  Figure 4. Empagliflozin (Empa) attenuated adipose tissue and liver inflammation in high-fat diet (HFD)-fed mice. (A) F4/80 immunostaining in the epididymal white adipose tissue (eWAT). Scale bars=100 µm. (B) Levels of F4/80 and inflammatory cytokines and chemokines mRNAs in the eWAT. (C) Immunoblots of phosphorylated nuclear factor-κB (p-NF-κB p65), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), phosphorylated extracellular signal-related kinase (p-ERK) and their total protein contents in the eWAT. (D) Levels of adiponectin mRNAs in the eWAT and adiponectin in the plasma. (E) F4/80 immunostaining in the liver. Scale bars=100 µm. (F) Levels of F4/80 and inflammatory cytokines and chemokines mRNAs in the liver. (G) Immunoblots of p-NF-κB p65, p-p38 MAPK and p-ERK, and their total protein contents in the liver. (H) Plasma interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1 and IL-33 contents. Data are presented as means±SEM, n=7–8. *P<0.05 and **p<0.01, respectively, for comparisons with normal chow (NC)-fed mice; #p<0.05 and ##p<0.01, respectively, for comparisons with HFD-fed mice.

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  Figure 5. Empagliflozin (Empa) promoted dominance of M2 over M1 macrophages in the epididymal white adipose tissue (eWAT) and liver. (A) Representative plots of total macrophages in the eWAT. (B) Occurrence frequencies of M1-type and M2-type macrophages in the eWAT. (C) Quantification of total macrophages, percentages of M1-type and M2-type macrophages and M1/M2 macrophage ratios in the eWAT. (D) Representative plot of total macrophages in liver. (E) Occurrence frequencies of M1-type and M2-type macrophages in the liver. (F) Quantification of total macrophages, percentages of M1-type and M2-type macrophages, and M1/M2 macrophage ratios in the liver. Data are presented as means±SEM, n=7–8. *P<0.05 and **p<0.01, respectively, for comparisons with normal chow (NC)-fed mice; #p<0.05 and ##p<0.01, respectively, for comparisons with high-fat diet (HFD)-fed mice. ATM, adipose tissue macrophage.